N-Nitroso Alkylating Agents: Formation and Persistence of Alkyl Derivatives in Mammalian Nucleic Acids as Contributing Factors in Carcinogenesis&lt;xref ref-type="fn" rid="FN5"&gt;5&lt;/xref&gt;
Journal: JNCI Journal of the National Cancer Institute (1979)

Many alkylating agents, either directly acting or only able to alkylate upon metabolic activation, show varying activity as carcinogens.The same agents also show varying activity as mutagens.Inasmuch as mutagenic reactions of DNA are likely to lead to genetic changes that could cause neoplastic transformation, the question arose as to whether the differences in reported carcinogenicity of alkylating agents could be due to differences in their chemical reactions with nucleic acids.Other types of carcinogens that are only reactive when metabolically activated differ from simple alkylating agents in both the size and position of substituents on nucleic acids.Some comparisons are made of the biologic effects of bulky substituents as compared to small alkyl groups. HISTORICAL PERSPECTIVEUntil the late 1960's it was generally believed that alkylation of the N-7 of G was the biologically important event.The foundation for this assumption was based on studies with such alkylating agents as sulfur mustard, dimethyl sulfate, and methyl methanesulfonate.These classes of directly acting agents that do react primarily at the N-7 of G are, however, not particularly carcinogenic (1).An important clue that ultimately led to new findings was that ethyl methanesulfonate was much more carcinogenic in rats than methyl methanesulfonate, although the absolute amount of N-7 ethylguanine in DNA of various tissues was only a small fraction of the amount of N-7 methylguanine when the animals were treated with the respective alkylating agents under the same conditions (2).It thus appeared likely that carcinogenesis did not result only, or primarily, from reaction at the N-7 of G.We therefore initiated comparative studies of the sites of reaction of a number of methylating and ethylating agents acting on polynucleotides.We were able to show that the simple ethylating agents acting in neutral aqueous solution on polynucleotides modified the N 4 of C and the N of A, whereas the analogous methyl derivatives were not detected (3,4).This demonstration lent support to the concept that methylation and ethylation could differ markedly in their affinities for specific sites.It could also be shown that, in addition to the nature of the alkyl group affecting the site of alkylation, the reactions of N-nitroso alkylating agents differed from those of the types of reagents first studied.The particular and unusual reactivity of N-nitroso alkyl compounds was first elucidated by us in 1968 while working with tobacco mosaic virus and its RNA.Thus N-methyl-N'-nitro-N-nitrosoguanidine, recognized at that time as a powerful mutagen and carcinogen, was demonstrated to cause methylation of nucleic acids (5) but to have markedly different site affinities and conformation dependence than the classical methylating agents (6).The interest in N-nitroso compounds was further stimulated when in 1970 Lawley and Thatcher (7) found 06-MeG, first detected by Loveless (8), to be present in N-methyl-N'-nitro-N-nitrosoguanidine-treated nucleic acids but not in dimethyl sulfatetreated nucleic acids.This was perhaps the first indication that some methylating agents could also react at sites other than those of the ring nitrogens.The difference between N-methyl-N'-nitro-nitrosoguanidine and dimethyl sulfate was also evident in their biologic effects.N-Methyl-i\T'-nitro-N-nitrosoguanidine is a powerful carcinogen, whereas dimethyl sulfate is a very weak carcinogen.Further work by Goth and Rajewsky ( 9) and Lawley and Warren (10) established that a carcinogenic Nnitroso ethylating agent, ethylnitrosourea, reacted in vivo to a relatively greater extent at the 0 6 of G than did methylnitrosourea, as reponed by Kleihues and colleagues (11,12), Walker and Ewart (13), and Frei and Lawley (14).However, just as with the alkyl ABBREVIATIONS USED: The four base moieties in a polynucleotide or nucleic acid are abbreviated with the use of a single letter; A, G, U, or T and C stand for adenine, guanine, uracil, or thymine and cytosine, respectively.Methyl and ethyl are abbreviated Me and Et, respectively.Thus 7-EtG refers to the product of ethylation of the N-7 of guanosine in a nucleic acid and not to reaction of the isolated base.